Antigenic variation plays a vital role in the pathogenesis of many infectious agents, most notably bacteria and protozoa that cause chronic infections. Borrelia burgdorferi and related organisms cause Lyme disease, a multistage, systemic infection that is transmitted by Ixodes ticks and has dermatologic, neurologic, arthralgic, and constitutional manifestations in humans and other animals. Lyme disease Borrelia have an elaborate antigenic variation system consisting of vlsE, a gene expressing a surface-exposed lipoprotein, and a series of vls silent cassettes, which serve as the source of sequences for random, segmental gene conversion events that change the sequence of the central cassette region of vlsE. It has been established that vlsE expression and variation are required for persistence of infection and immune evasion, yet the mechanisms that govern this biologically important process are poorly understood. In this project, several approaches will be used to address these questions. In Specific Aim 1, an in vitro model system for studying vlsE recombination will be developed. vlsE expression and variation will be analyzed in vivo by qPCR and sequence specific PCR. Deficiencies of vlsE expression and variation and their effect on B. burgdorferi infectivity will be examined using knockdown constructs and mutant strains deficient in vlsE variation. Specific Aim 2 explores the mechanism of vlsE recombination. DNA repair and recombination mutants and mutants with 'vls minus'phenotypes will be examined for deficiencies in vlsE recombination. Expression libraries will be used to screen for factors that regulate vlsE expression and variation. Finally the role of anti-VlsE antibodies in mediating clearance of variant and non-variant organisms will be determined in Specific Aim 3 through passive and active immunization studies. These studies will define the functional roles of VlsE in immune evasion and identify the counteracting anti-VlsE antibody responses and their efficacy in immune clearance.